A body of an animal is composed of combination of four kinds of tissue types called an epithelial tissue, a connective tissue, a muscle tissue and a nervous tissue, and the epithelial tissue or the muscle tissue is mainly composed of a cell, while a main constituent of the connective tissue is an extracellular matrix called stroma having a collagen fiber as a main component. In addition, there is present a sheet-like extracellular matrix called a basement membrane between the epithelial tissue and connective tissue, and a cell composing the muscle tissue is also covered with the basement membrane at the periphery, and a blood vessel is also backed with the basement membrane in an endothelial cell thereof. Further, most of cells composing a multicellular organism have the basement membrane as an anchorage thereof, such that a nervous cell or the Schwann cell enveloping, for example, an axon of a peripheral nerve also has the basement membrane as the anchorage thereof, or the like. The extracellular matrix is required for growth or survival of a cell; for example, an animal cell not only cannot grow but also self-destructs with generating apoptosis, unless an animal cell can adhere to a culture dish and form the anchorage. This phenomenon is called anchorage dependence of cell growth, and this phenomenon is observed in almost all cells composing the multicellular organism, except a malignant cancer cell or a blood corpuscle cell.
The basement membrane to be the anchorage in such cell culture is the extremely thin sheet-like extracellular matrix with a thickness of about 100 nm, and is composed of a Type IV collagen, a laminin, a nidogen, a heparan sulfate proteoglycan or the like, which compose a three-dimensional structure by binding in a complex way. For example, the Type IV collagen is a main constituent bearing morphology maintenance of the basement membrane, and forms a tetramer cross-linked by a disulfide bond at an N-terminal region, while associates in an end-to-end type at a spherical region of a C-terminal, thus constructs a two-dimensional network structure through this association of N-terminal region and the C-terminal region. In addition, the laminin is a hetero trimer molecule, where three subunit chains called α, β and γ are associated, and is a component of the basement membrane having a cross shape. There are present 11 kinds of subunits in total, wherein there are present α1 to α5 in the α-chain, β1 to β3 in the β chain, and γ1 to γ3 in the γ chain. There have been known 15 kinds of laminins at present time by a combination of these subunits, names thereof are called by a constitution of the subunit chains. The three subunit chains have a common coiled coil domain to form the hetero trimer by association in this region. On the other hand, the N-terminal region of each subunit has affinity mutually, and is self-organized to the basement membrane by association in this region. It should be noted that laminin self-associated at the base surface acquires physical strength, by bonding with the Type IV collagen via nidogen and being backed with the network structure, which the Type IV collagen creates independently by self-association. Further, the structure thereof is stabilized by adding heparan sulfate proteoglycan, and by mutual action between the basement membrane molecules, and an intermolecular cross-linking by a disulfide bonding or a non-disulfide bonding.
Conventionally, research on the extracellular matrix has been carried out mainly on a collagen. In the mammals, about 30 kinds of genetically different collagens have been found, and they are named a Type I, a Type II, a Type III or the like in the order of discovery. Each of them differs in a structure, function and distribution, however, it has a right-handed helical structure, where three polypeptide chains called the α-chain are wound mutually, and the polypeptide chain is common in having a basic structure composed of repetition of Gly-X—Y (wherein X and Y represent an arbitrary amino acid). It should be noted that in the Type I collagen, telopeptide, which is a non-helical region, is present at the both ends of the three-helical region.
A major collagen composing the basement membrane is the Type IV collagen. It forms the helical structure of the three chains similarly as the Type I collagen, however, it differs from the Type I collagen in that it has a part, where the Gly-X—Y structure interrupts. In addition, the Type IV collagen has a 7S region containing many cysteine residual groups at the N terminal, and a non-collagen helical region (NC1 region) at the molecular C terminal, which are important regions in that the two-dimensional network structure of the Type IV collagen is formed via the 7S region and the NC1 region.
In general, a collagen of the connective tissue of an animal is easily extracted by heat treatment, however, in this case, the specific three-helical structure thereof is destructed by thermal denaturation of the collagen to become a gelatin state. A collagen is generally poorly soluble in a state that the stereo structure thereof is maintained as it is, and a particular method becomes necessary to obtain the collagen as a solution. As an extraction method for the Type IV collagen, there have been known a method for extracting the Type I, III, V and IV collagens contained in a placenta by pepsin treatment using the placenta as a material, and then purifying the Type IV collagen by salt fractionation, a column or the like (NON-PATENT LITERATURE 1: Sage H, etal. J. Biol. Chem. 254, 9893-9900 (1979)); or a method for extracting the Type IV collagen in a non-enzymatic way in an acidic solution from a lens capsule of an animal eyeball only composed of almost the Type IV (NON-PATENT LITERATURE 2: Muraoka, M and Hayashi, T., J. Biochem. 114, 358-362 (1993)).
On the other hand, among various collagens, for example, the Type I collagen is a major constituent of the connective tissue such as skin, tendon, bone or the like of an animal, having a helical structure of about 300 nm, where three polypeptide chains are bound mutually. A slender fiber is created by association of these three-helical bodies in mutually shifting by 1/4.4, which further forms a bundle by alignment in parallel, and forms a collagen fiber which endures against strong tension. As an extraction method for the Type I collagen, there has been known an extraction method with an acidic solution or by enzyme treatment using bone or skin or the like of an animal as a material (PATENT LITERATURE 1: JP-B-37-14426); or a method for solubilization by alkali treatment (NON-PATENT LITERATURE 3: Fujii, T. Hoppe-Seyler's Z. Physiol., Chem. 350, 1257-1265 (1969)) or the like.
The Type I collagen or Type IV collagen is a constituent of the extracellular matrix, and acts as a cell adhesion factor. This is the reason for carrying out collagen coating of the cell culture plate in cell culture. On the other hand, the Type I collagen or Type IV collagen differs in fibrosis ability or gel forming ability or others depending on the extraction method thereof, and shape of the collagen in the coated layer differs depending of the collagen coating method.
There is a report that, although cell doubling time was same in the collagen coated dish and the three-dimensional collagen fiber gel, extension by 1.5 times and significant growth inhibition were observed in a cell-containing three-dimensional collagen gel, when a fibroblast was cultured using three kinds of the cell culture plates, that is, one (a collagen coated dish) obtained by putting a hydrochloric acid solution of the Type I collagen treated with pepsin on the surface of the cell culture plate, and drying with sterile air flow at 25° C.; one (the three-dimensional collagen fiber gel) obtained by stirring the hydrochloric acid solution of collagen with a DMEM culture medium containing penicillin, streptomycin, FBS-containing DMEM culture medium, and putting this on the cell culture plate and incubating this at 37° C. for 6 hours under CO2 condition; and one (the cell-containing three-dimensional collagen gel) obtained by mixing the collagen culture medium solution and a cell suspension solution, and immobilizing onto the cell culture plate(NON-PATENT LITERATURE 4: Nishiyama, T. et al. Matrix, 9, 193-199 (1989)).
In addition, apoptosis is induced in a fibrous collagen, when a keratinocyte was cultured by the non-fibrous collagen obtained by coating a phosphoric acid buffer solution not containing calcium and magnesium of the Type I collagen onto the cell culture plate, and mounting at room temperature for 2 hours, and by the fibrous collagen obtained by putting a PBS (−) solution of the Type I collagen onto the cell culture plate and incubating at 37° C. for 2 hours to associate in a fibrous form (NON-PATENT LITERATURE 5: Fujisaki, H. and Hattori, S., Exp. Cell. Res. 280, 255-269 (2002), NON-PATENT LITERATURE 6: Fujisaki, H. et al. Connect. Tissue Res. 48, 159-169 (2007)).
It should be noted that there is “Matrigel” as the cell culture plate coated with a basement membrane component. The “Matrigel” is composed of an extract of sarcoma, which excessively produces a basement membrane molecule called EHS (Engelbreth-Holm-Swarm) sarcoma of a mouse, and further, is one blended with laminin, heparan sulfate proteoglycan, entactin or the like.